Alkylating agents, such as nitrogen mustards, have been used in the pharmaceutical industry as anti-cancer drugs. For example, nitrogen mustards have been used to treat cutaneous T-cell lymphoma (CTCL), including mycosis fungoides (MF).
CTCL is a cancer of the white blood cells that primarily affects the skin and only secondarily affects other sites. The disease involves the uncontrolled proliferation of T-lymphocytes known as T-helper (CD4+) cells of the immune system. The proliferation of T-helper cells results in the penetration, or infiltration, of these abnormal cells into the epidermal layer of the skin. The skin reacts with slightly scaling lesions that itch, although the sites of greatest infiltration do not necessarily correspond to the sites of the lesions. The lesions are most often located on the trunk, but can be present on any part of the body. In the most common course of the disease, the patchy lesions progress to palpable plaques that are deeper red and have more defined edges. As the disease worsens, skin tumors develop that are often mushroom-shaped, hence the name mycosis fungoides. Finally, the cancer progresses to extracutanous involvement, often in the lymph nodes or the viscera.
CTCL is a rare disease, with an annual incidence of about 0.29 cases per 100,000 persons in the United States. It is about half as common in Eastern Europe. However, this discrepancy may be attributed to a differing physician awareness of the disease rather than a true difference in occurrence. In the United States, there are about 500-600 new cases a year and about 100-200 deaths. CTCL is usually seen in older adults; the median age at diagnosis is 55-60 years. It strikes twice as many men as women. The average life expectancy at diagnosis is 7-10 years, even without treatment.
Alkylating agents, such as nitrogen mustards, are believed to have anti-cancer activity by acting on the nucleic acids of DNA and RNA. Alkylating agents have four main actions on nucleic acids. First, the agents may cause crosslinking of DNA strands which interferes with DNA and RNA synthesis. This is thought to be the most important reason for the cytotoxic effect of alkylating agents. Secondly, the agents may alter the “side chain groups” of the nucleotide base ring which would lead to abnormal base pairing and point mutations in the synthesized DNA and RNA chains. Thirdly, the alkylating agents may split the base ring from the nucleotide which again interrupts proper DNA and RNA synthesis. Finally, the alkylating agents may break the ring structure of a nucleotide base which would prevent base pairing during DNA and RNA synthesis.
Nitrogen mustards are believed to act as anti-cancer agents by impairing natural DNA strand replication of cancer cells. In natural DNA strand replication, a DNA strand having deoxyribonucleosides, wherein each deoxyribonucleoside may include a base adenine (A), thymine (T), cytosine (C) and guanine (G), replicates by linking each deoxyribonucleoside on the strand with another deoxyribonucleoside, wherein typical linking occurs between adenine (A) and thymine (T), forming an A-T linkage, and between cytosine (C) and guanine (G), forming a C-G linkage, between the original DNA strand and its replicated DNA strand. Nitrogen mustards are believed to allow unnatural base-base linkages such as a guanine (G) base linking to another guanine (G) base if the particular nitrogen mustard is a bifunctional alkylator. As used herein, unless otherwise defined, a bifunctional alkylator is a nitrogen mustard that has at least two 2-chloroethyl side chains, for example, bis-(2-chloroethyl)methyl amine.
Nitrogen mustards may allow unnatural base-base linkages in DNA, for example, by the mechanism depicted in Reactions 1 to 4 below.
First, a nitrogen mustard, such as bis-(2-chloroethyl)methylamine (I) undergoes an intramolecular cyclization, resulting in formation of a highly reactive ethyleniminium intermediate (i.e., a aziridinium cation) (II) according to the following Reaction 1.

In the bis-(2-chloroethyl)methylamine (I), a carbon atom bonded to chlorine may initially have a partial positive charge, δ+, and a chlorine atom may initially have a partial negative charge, δ−. In Reaction 1, an unshared pair of electrons of nitrogen may form a covalent bond to the carbon having δ+, releasing the chlorine atom as chloride, and forming the ethyleniminium intermediate (II). A concentration of the ethyleniminium intermediate (II) may be in equilibrium with a concentration of the bis-(2-chloroethyl)methylamine (I) wherein the equilibrium constant Keq(1a,1b) may be represented by a ratio of a rate k1a, of the forward reaction 1a, to a rate k1b, of the reverse reaction 1b.
Second, the ethyleniminium intermediate (II) formed in Reaction 1 undergoes nucleophilic attack by an electron donor (i.e., a nucleophile, such as the guanine (III) of DNA), whereby the nucleophile is alkylated to form alkylated deoxyribonucleoside (IV) according to the following Reaction 2.

Reaction with the nucleophile guanine (III) at the position N-7 of the guanine occurs to the greatest extent. Other sites on guanine, and other DNA bases, such as adenine, cytosine, and thymine, and phosphate oxygens can also be alkyated.
Third, the alkylchloroethyl side chain of the alkylated deoxyribonucleoside (IV) formed in Reaction 2 undergoes intramolecular cyclization, resulting in formation of deoxyribonuceloside having a highly reactive aziridinium ring (V) according to the following Reaction 3.

Finally, another guanine (III) of DNA reacts with the deoxyribonucleoside having a highly reactive aziridinium ring (V) formed in Reaction 3 to form an unnatural guanine-guanine link between two strands of DNA, as depicted in Structure (VI), according to the following Reaction 4.
However, only the species which have the potential to form DNA cross-links, i.e. bifunctional species (I and II, Reactions 1-2) are believed to be the active forms of the nitrogen mustard alkylating agents;
The electrophilicity of alkylating agents, such as nitrogen mustards, causes them to be subject to decomposition in the presence of natural nucleophiles in the environment, such as water. In this work HPLC has been employed to determine the degradation products of mechlorethamine in ointment. The degradation (hydrolysis) of mechlorethamine is well characterized giving rise to N-methyl ethanolamine. Alternatively, the drug can degrade by reacting with a wide variety of nucleophiles to form covalent adducts.
Thus, there is a need in the art for stable compositions of alkylating agents, such as nitrogen mustards, that are suitable for topical use.